Rare histologic transformation of a CTNNB1 (β-catenin) mutated prostate cancer with aggressive clinical course

Background Catenin (Cadherin-Associated Protein), Beta 1 (CTNNB1) genomic alterations are rare in prostate cancer (PCa). Gain-of-function mutations lead to overexpression of β-catenin, with consequent hyperactivation of the Wnt/β-catenin signaling pathway, implicated in PCa progression and treatment resistance. To date, successful targeted treatment options for Wnt/β-catenin - driven PCa are lacking. Methods We report a rare histologic transformation of a CTNNB1 (β-catenin) mutated metastatic castration resistant prostate cancer (mCRPC), clinically characterized by highly aggressive disease course. We histologically and molecularly characterized the liver metastatic tumor samples, as well as successfully generated patient-derived organoids (PDOs) and patient-derived xenograft (PDX) from a liver metastasis. We used the generated cell models for further molecular characterization and drug response assays. Results Immunohistochemistry of liver metastatic biopsies and PDX tumor showed lack of expression of typical PCa (e.g., AR, PSA, PSAP, ERG) or neuroendocrine markers (synaptophysin), compatible with double-negative CRPC, but was positive for nuclear β-catenin expression, keratin 7 and 34βE12. ERG rearrangement was confirmed by fluorescent in situ hybridization (FISH). Drug response assays confirmed, in line with the clinical disease course, lack of sensitivity to common drugs used in mCRPC (e.g., enzalutamide, docetaxel). The casein kinase 1 (CK1) inhibitor IC261 and the tankyrase 1/2 inhibitor G700-LK showed modest activity. Moreover, despite harbouring a CTNNB1 mutation, PDOs were largely insensitive to SMARCA2/4- targeting PROTAC degraders and inhibitor. Conclusions The reported CTNNB1-mutated mCRPC case highlights the potential challenges of double-negative CRPC diagnosis and underlines the relevance of further translational research to enable successful targeted treatment of rare molecular subtypes of mCRPC. Supplementary Information The online version contains supplementary material available at 10.1186/s13000-024-01511-3.


Patient-derived xenograft (PDX)
PDOs from passage 6 were used for PDX generation.1.8 × 10 6 viable cells were injected subcutaneously (sc) in a 2-weeks old NOD scid gamma (NSG) male mouse.The mouse was regularly assessed for sc tumor growth (initially once weekly, and 2 times/week from detection of palpable tumor).Tumor would be allowed to grow to a maximum of 1 cm 3 .All experiments were performed in agreement with local laws and regulations.

DNA sequencing
DNA was extracted from fresh-frozen liver metastatic tumor tissue with using the AllPrep DNA/RNA FFPE kit (Qiagen, catalog 80,234), and DNA concentration measured with Qubit (Thermo Fischer Scientific).Hybrid capture-based next-generation sequencing (NGS) was performed using the commercially available Foundation-One® CDx assay, which interrogates 324 genes for substitutions, indels and copy number variations, and 36 genes for rearrangements, as well as provides information on tumor mutational burden (TMB) and microsatellite instability status [18].

Fluorescent in situ hybridization (FISH)
FISH for the detection of the TMPRSS2/ERG rearrangement was performed at the Institute of Pathology, University Hospital Basel, Switzerland, using the commercially available ZytoLight® ERG-dual color break apart probe (ZytoVision GmbH, Bremerhaven, Germany, catalog Z-2138-200), according to the manufacturer's recommendations.Signals were counted in at least 50 tumour nuclei using an epifluorescence microscope (Axioplan 2 Imaging; Carl Zeiss, Oberkochen, Germany).FISH for ERG rearrangement was defined by the loss of one green signal (5′ probe) or as a separate green, and a separate orange signal in at least 15% of analyzed tumor cells.

Clinical presentation
An 82-year-old patient was diagnosed with de novo metastatic hormone-sensitive prostate cancer.His previous medical history included hypertension, dyslipidemia, and atrial fibrillation.Biopsy of the prostate revealed an adenocarcinoma Gleason score (GS) 4 + 4 = 8 with pronounced cribriform growth pattern without neuroendocrine differentiation.The prostate-specific antigen (PSA) value at presentation was 304 µg/l.Conventional imaging with computer tomography (CT) of chest-abdomen and bone scan showed pelvic and abdominal lymph node metastases (M1a), with no signs of visceral or bone involvement.First-line treatment with a gonadotropinreleasing hormone (GnRH) analogue and apalutamide was initiated.Radiotherapy to the prostate given low volume disease analog STAMPEDE trial was evaluated but rejected by the patient [19].Under first-line treatment with GnRH and apalutamide, the PSA value dropped down to 0.04 µg/l.Two years after treatment initiation the patient reported intense fatigue, dysuria, and peripheral edema of the lower limbs.Laboratory values revealed a grade 1 increase of transaminases, an alkaline phosphatase of 288U/l and a lactate dehydrogenase of 646 U/l, as well as a low PSA (0.1 µg/l) and elevated neuronspecific enolase values (236 µg/l).CT imaging revealed local progression of the primary tumor infiltrating bladder and rectum and new extensive metastatic spread to the lung and liver.Liver biopsy was performed to assess small-cell transformation, revealing a highly proliferative high-grade adenocarcinoma with prominent nucleoli; no neuroendocrine features were observed.Based on the working current classification of advanced PCa, this tumor fits into a class of double-negative CRPC expressing neither AR or neuroendocrine features (personal communication PCF Pathology working group).The patient presented rapid clinical deterioration complicated with sepsis and leading to death one month later (Fig. 1).

Phenotypic and genomic characterization
While primary PCa tumor showed mucin-containing glandular formation, liver biopsy and PDX hematoxylin and eosin (H&E) slides showed high-grade solid carcinoma with prominent nucleoli, lack of cribriform growth pattern, absence of acid mucin and no evidence of neuroendocrine features (Fig. 2).The liver metastases and PDX tumor histology slides were negative for AR, PSA, PSAP and ERG protein expression; the tumor strongly expressed KRT7 and focally 34βE12 (Fig. 3).To help exclude a urothelial carcinoma keratin 34βE12 was performed, resulting in focal positivity (Fig. 3A-B) [20].β-catenin IHC nuclear staining was strongly positive in the derived PDX (UB_PCa03), as well as in the established Wnt-dependent PCa PDO WCM1078 [10] (Fig. 4A-B).On the contrary, in the neuroendocrine PDO PM154, β-catenin staining was uniquely cytoplasmatic, acting as a negative control (Fig. 4C).Targeted DNA NGS (FoundationOne®CDx) performed from the liver metastatic biopsy material uncovered a hotspot CTNNB1 gain-of-function mutation in exon 2 (94G > A, D32N), as well as a loss-of-function mutation in TP53 (R282W), along with PTEN loss and a TMPRSS2 rearrangement (with unclear partner).The tumor molecular burden was low (2.4 mutations/megabase) and the microsatellite status stable (Fig. 5A, Suppl.Table 1).The presence of an ERG rearrangement was confirmed by FISH on the PDX histology slides, showing a split signal in 92% of the analyzed tumor cells (Fig. 5B).

Patient-derived organoids and xenografts
3D PDOs (UB_PCa03) were derived from fresh liver metastatic tumor tissue, following the protocol described in the Methods section, and could be successfully used for drug response assays (Fig. 6A).Por PDX derivation, 1.8 × 10 6 viable cells (passage 6), were sc injected in an NSG male mouse.Palpable sc tumor was first detected at day + 16, reaching maximum volume at day + 35 (Fig. 6B-D).

Discussion
We report a rare transformation of a metastatic PCa, acquiring histological features of urothelial carcinoma, atypical IHC pattern and a highly aggressive disease course.However, DNA NGS from liver metastatic biopsies revealed, along with the clonal CTNNB1 gain-of-function mutation (D32N), molecular findings characteristic for PCa, such as the presence of a TMPRSS2 rearrangement, a loss-of-function mutation in TP53 (R282W), and PTEN loss.FISH performed on PDX tumor material could confirm the presence of the TMPRSS2: ERG rearrangement, reassuring the final diagnosis of PCa metastases.In line with patient aggressive clinical disease course, derived PDOs showed lack of sensitivity to common drugs used for mCRPC, such as enzalutamide and docetaxel.We further assessed sensitivity to several experimental drugs with potential activity in tumors with Wnt/β-catenin pathway activation.Our preliminary drug response results showed only modest sensitivity to treatment with the CK1 inhibitor IC261 and the tankyrase inhibitor G007-LK, which are known to interfere with Wnt/β-catenin signaling in PCa [7].Further experiments would be required to confirm these findings.CK1α phosphorylates β-catenin and induces its degradation, so that inhibition of CK1α leads to an excessive Wnt/β-catenin activation 3,29 .Moreover, both CK1α and CK1δ phosphorylate the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), resulting again in Wnt/β-catenin pathway activation [29,30].IC261 inhibits CK1δ, CK1ε and CK1α, therefore, having potential impact on the WNT/β-catenin signaling.However, further work showed that IC261 may induce Wnt-independent cancer cell death, contrary to other CK1δ/ε inhibitors [22].Tankyrase 1/2 inhibitors lead to decreased degradation of axin, a negative regulator of the Wnt/β-catenin promoting β-catenin degradation [31].Based on previous data showing that BRG1 (SMARCA4), a key component of the SWI/SNF chromatin remodeling complex, plays a relevant role in the Wnt//β-catenin pathway regulation, we additionally aimed to assess sensitivity to SMARCA2 and SMARCA4 PROTAC degraders and inhibitors [16,[32][33][34].Moreover, increased Wnt//β-catenin signaling activity has been demonstrated in PCa tumors with high SMARCA4 levels [35].However, none of the used drugs targeting the SWI/SNF complex components SMARCA4 and SMARCA2 showed activity in the reported PDO.
Finally, the morphologic and molecular features of this double negative PCa highlight a challenge in the classification of advanced metastatic CRPC.Histology alone, unlike primary untreated PCa, is insufficient to adequately characterize these tumors.A recent working group on the pathology of CRPC is supporting a classification approach that includes morphology, molecular, and IHC information (Hafner, Rubin, Beltran, in preparation).For example, in this case high-grade adenocarcinoma would be modified with the description of double negative CRPC referring to the lack of AR activity or neuroendocrine features; these features may only become

Conclusions
We characterized a rare histologic transformation of an advanced CTNNB1-mutated mCRPC, clinically characterized by an aggressive lung and hepatic metastatic spread and fulminant disease course.We successfully derived PDOs and PDX from liver metastatic tumor material.Derived PDOs showed, as expected, lack of response to common drugs used in mCRPC, such as enzalutamide and docetaxel.Preliminary experiments showed only a modest sensitivity to the CK1 inhibitor IC261 and to the tankyrase inhibitor G007-LK, which are known to interfere with Wnt/β-catenin signaling.Further research work is needed to explore the effect of these compounds in more detail.The reported mCRPC case underlines the need of further research work required to enable successful targeted treatment of rare molecular subtypes of PCa.

Fig. 1
Fig. 1 Schematic illustration of patient's clinical disease course.(A) Timeline showing disease course and received treatment lines; (B) PSA levels over disease course; (C) Computed tomography image of liver metastatic spread (yellow arrow shows liver metastases).GnRH: gonadotropin-releasing hormone; mHSPC: metastatic hormone-sensitive prostate cancer; PSA: prostate-specific antigen

Fig. 2
Fig. 2 Hematoxylin and eosin (H&E) staining of (A) primary PCa tumor, (B) liver metastases and (C) patient-derived xenograft (PDX) tumor material.(A) Primary PCa tumor showing glandular formations with mucin; (B) Liver metastasis showing highly proliferative high-grade adenocarcinoma with prominent nucleoli; no neuroendocrine features are observed; (C) PDX tumor material showing highly proliferative neoplasia, histologically similar to the liver metastatic biopsy